Haemostasis or stoppage of blood flow can be shown to be a disturbance of a delicately poised system of two processes--coagulation and fibrinolysis. Under normal circumstances blood remains fluid, but if vascular damage occurs or if certain abnormal physiological states develop, steady states in one or both of these processes are disturbed and haemostasis results.
Blood coagulation involves more than 50 important substances which are found in the blood and tissues, some promoting coagulation ("procoagulants"), and others inhibiting coagulation ("anticoagulants"). Whether or not blood coagulates depends on the degree of balance between these two groups of substances. In the healthy individual, the anticoagulants normally predominate, and the blood remains fluid. In the stressed individual, that is those individuals with endogenously damaged vessels and especially those having certain abnormal physiological conditions, procoagulants in the affected area become "activated" and override the anticoagulants leading to the formation of thrombin which, in turn, leads to the development of a blood clot or thrombus. A thrombus is an aggregate of blood fractions, primarily platelets and fibrin with entrapment of cellular elements, frequently causing obstruction at the point of its formation.
There is general agreement that blood coagulation or clotting takes place in three essential steps. First, a complex of substances called prothrombin activator is formed, e.g., in response to rupture of the blood vessel or damage to the blood itself. Second, the prothrombin activator catalyses the conversion of prothrombin to thrombin. Third, the thrombin acts as an enzyme to activate platelets and to convert fibrinogen into fibrin threads that enmesh platelets, blood cells, and plasma to form the clot itself.
Platelets play a very important role in blood coagulation. Their role is twofold, they form aggregates and they provide procoagulant phospholipids, that is, negatively charged phospholipids. The aggregates serve as an initial plug with two functions, one which can prevent bleeding for a short period of time, and the other where they act as a sponge or niche of non-flowing plasma where thrombin can accumulate. This accumulated thrombin, in turn, activates the clotting mechanism in various ways, but importantly, it also activates platelets.
Thrombin is formed by activation of prothrombin with factor X.sub.a. Factor X.sub.a is formed by activation of factor X with factor IX.sub.a. Both activation reactions are slow in the absence of procoagulant phospholipids. A phospholipid membrane will only be procoagulant when a sufficient amount of negatively charged phospholipids (mostly phosphatidyl serene) are present (see, e.g., Bevers et al., Eur. J. Biochem. 122:429-436 (1982), the disclosure of which is hereby incorporated by reference). The outer leaflet of a resting platelet contains hardly any phosphatidyl serine. Thus, the membrane is hardly or not procoagulant. On activation of the platelet, the phosphatidyl serine present in the inner leaflet of the membrane will be exposed in the outer leaflet. This is the so-called flip-flop reaction and by this process the platelet becomes procoagulant.
Platelets can be activated not only by the natural activators thrombin and collagen, but also by calcium ionophore A23187 (Bevers, et al., supra), diamide (Van Rijn et al., Eur. J. Biochem. 133:1-10 (1983)) and several other compounds such as serotonin (Zucker and Nachtmias, Arteriosclerosis 5:2-18 (1985)), epinephrin, platelet activating factor, adenosine diphosphate, etc. (see Rapaport, Introduction to haematology:440-448). Because platelets are activated by thrombin, this compound facilitates its own formation. Besides procoagulant phospholipids, the cofactors, factors V.sub.a and VIII.sub.a, are required for optimal activation of prothrombin and factor X, respectively. These cofactors are formed by activation of factors V and VIII with trace amounts of thrombin. So also in this way thrombin promotes its own formation. How the first few molecules of factors V and VIII are activated is still a matter of speculation.
As noted above, the level of activated clotting factors in whole blood usually is low because all kinds of plasma inhibitors inactivate these clotting factors. Below a certain threshold these activated factors are not harmful. Also, the amount of procoagulant phospholipids in whole blood is low because resting platelets have a mechanism to transport phosphatidyl serine from the outer to the inner leaflet of the membrane. A minor amount of the phosphatidyl serine is probably still present in the outer leaflet causing a residual procoagulant activity of the platelets. This residual or "resting activity" establishes the threshold at which activated clotting factors may result in thrombosis. Thus, the susceptibility of an individual to get thrombosis may very well be correlated with the level of procoagulant activity of his platelets.
The formation of thrombi in the human has always been a clinical condition and thus a matter of diagnosis of an illness and treatment (see Rapaport, Introduction to Haematology:558-576).
Previous methods for assaying the formation of thrombin are in the great majority of cases an estimation of clotting times such as the prothrombin time in any of its multiple variations. These give no information on the procoagulant activity of platelets because external phospholipids are added. The whole blood clotting time shows a very large experimental error and is dependent on haematocrit clotting factors, platelets and fibrinolysis all at the same time. Specialized laboratory tests like the thrombin generation test in platelet rich plasma are more precise, but take at least half an hour of skilled laboratory personal and are not suitable for screening a population or hospital routine.
Thus, it would be desirable to establish a method for determining the procoagulant activity of resting platelets based on the availability of negatively charged phospholipids in the outer membrane of platelets. This would facilitate the establishment of a threshold above which it could be predicted that there is a risk of thrombosis occurring. Moreover, such a test would also be useful in evaluating the susceptibility of platelets to the activating action of thrombin. It has been found, for example, that some platelets are more susceptible to the activating action of thrombin than others. This may be related to the membrane composition of the platelet, membrane fluidity or the presence of platelet inhibitors. Easily triggered platelets may result in a higher thrombosis risk, therefore warranting preventative therapeutic measures. It would also be desirable to have a method for screening drugs which, for example, inhibit the flip-flop effect of negatively charged phospholipids in the platelet's membrane. Such a test could be used to develop drugs for reducing the excitability of platelets, thus reducing the risk of thrombosis to the patient. Finally, it would be desirable for the medical/clinical practitioner to have a method for measuring the procoagulant phospholipids in whole blood, as the isolation of platelets is time consuming and not readily applicable in clinical use. Moreover, by the isolation of platelets a serious risk of platelet activation is present.